Apparatus for obtaining increased particle concentration for optical examination

ABSTRACT

The present invention relates to a method and apparatus for analyzing a blood or other biological fluid sample in a quiescent state, whereby particulate constituents of biological samples that contain sparse populations of interesting cellular species can be enumerated and inspected using an optical scanning instrument. Specifically, this invention relates to a method and apparatus for obtaining increased cellular or particulate concentrations within the use of said optical scanning method.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for analyzing ablood or other biological fluid sample in a quiescent state, wherebyparticulate constituents of biological samples that contain sparsepopulations of cellular species of interest can be enumerated andinspected using an optical scanning instrument. Specifically, thisinvention relates to a method and apparatus for obtaining an increasedcellular or particulate concentration within the use of said opticalscanning method.

BACKGROUND OF THE INVENTION

The formation of appropriate cellular or particulate layers for lateroptical examination is important to many fields. One of these fields ishematology, where several methods and devices have been described forobtaining clinically useful cell concentrations.

A method and apparatus for analyzing a blood or other biologic fluidsample in a quiescent state without the need for separate fluid streamspassing through the blood sample during the analysis is described inU.S. Ser. No. 09/248,135 filed Feb. 10, 1999, now U.S. Pat. No.6,123,184, and U.S. Ser. No. 09/249,721 filed Feb. 12, 1999, now U.S.Pat. No. 6,235,536. Although this method simplifies the analysisprocedure and yields the full compliment of CBC parameters it alsopossesses several disadvantages. One disadvantage of the apparatus isthat the concentration of cells in the examination layer is notcontrolled. This can lead to difficulties in optically examining cellvolume and morphology. Another disadvantage of the aforementionedapparatus is that the field of cells may be too sparse in clinicallyrelevant samples to complete scanning in a timely manner.

U.S. Pat. Nos. 5,627,041 and 5,912,134 describe an apparatus and methodfor cytometric measurement of cell populations using fluorescentmarkers. However, a disadvantage of this method is that, if the sampleunder test is blood, it requires addition of diluent in such quantitiesthat white blood cells (WBCs) with depressed counts are not verynumerous in the sample and may require extremely long examination timesto locate them. Moreover, if the cell counts within the undiluted bloodare low, clinically relevant cell populations present in the sample maynot be detected in the diluted sample. For example, patients who undergochemotherapy regimens may have depressed white cell counts in the rangeof 1000 cells/μL and less. Cytometric examinations are typicallysearching for a sub-population of these cells, further reducing thelikelihood of locating them.

U.S. Pat. No. 4,790,640 discloses a wedge shaped device for trappingrigid particles, such as sickle cells in blood. However, a disadvantageof the device is that the selection of cell sizes is accomplished bythickness of the chamber alone, which can exhibit substantialmanufacturing variation over the examination area, causing acorresponding loss of ability to separate by size.

Consequently, it would be desirable to have a method and apparatus forobtaining the desired cellular concentration in a blood or otherbiologic sample which can mitigate the effect of a separate dilutionstep and addition of diluting fluids.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide channels in aseparation wall inside a separation chamber, said channels havingappropriate size and dimensions to allow at least one undesired particlespecies to pass while excluding larger particles from passing through,thereby arriving at a predetermined increased volume fraction of thedesired particles.

It is another objective of the present invention to incorporate at leasttwo separating channels in a separation wall in a separation chamber,the channels having channel sizes selected to allow at least one cellspecies and the substantially liquid component of the sample to passthrough them, arriving at a desired concentration of larger cell typesin the first compartment in front of the separation wall. A furtherembodiment of the present invention is to have a plurality of separatingchannels in the separating wall having one channel size selected toallow at least one cell species and the substantially liquid componentof the sample to pass through them.

It is a further objective of the present invention to regulate thevolume fraction of cellular or particle components of a specimen bymeans of an array of separating channels which effect their selection bymeans of size exclusion during flow between two adjoining compartments.

It is another objective of the present invention to increase theconcentration of larger particles for cytometric examination of sparsepopulations by allowing only smaller particles and substantially liquidcomponents to pass through the channels into a subsequent chamber.

It is a still further objective of the present invention to create anaccurate spacing between two opposing containment walls to allow for theoptimal formation of desired regions where all particles of interest liein the same focal plane, allowing an accurate determination of thechamber thickness without relying on extraneous equipment andmanipulations for height calibration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an isometric view of the particle separation chamber with thelid removed showing the separation channels and adjacent cellexamination areas.

FIG. 2 is an isometric view of the bottom portion of the separationchamber showing the disposition of the separation channels.

FIG. 3 shows a detailed view of another embodiment of the separationchannels.

FIG. 4 shows an isometric view of another embodiment of the separationchannels.

FIG. 5 is a schematic view of a particle separation chamber withincreased analytical sensitivity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method and apparatus for obtaining anincreased cellular or particulate concentration in a substantiallydilute sample. The apparatus includes a sample chamber which hasopposing sample containment walls, at least one of which is transparent.The sample chamber is separated into adjoining compartments which are influid communication by means of a multiplicity of channels alignedsubstantially parallel to each other and which traverse a separatingwall between the two compartments. Filling of the chamber results in asubstantially quiescent sample ready for further examination.

In a preferred embodiment, a chamber is used to manipulate bloodcomponents and one type of channel in the array is of sufficient sizeand dimensions to allow red blood cells (RBCs) to pass while excludinglarger white blood cells (WBCs). The dimensions of the channel to createthe desired size exclusion are nominally, in a preferred embodiment, 3to 10 μm deep×5 to 50 μm wide. Dimensions within this range have beenselected to effect the desired volume flow rate of RBCs, orparticle-containing sample. In a modification of the invention, anothertype of channel in the array is of sufficient size and dimensions toexclude RBCs and WBCs from passing while allowing the liquid componentof the specimen to pass freely. The dimensions of the channel to createthe desired size exclusion are nominally, in a preferred embodiment, 0.5to 1.5 μm deep×50 to 1000 μm wide. Dimensions within this range areselected to effect the desired volume flow rate of the liquid-onlyportion of the sample.

As the blood sample or particle containing specimen flows from the firstcompartment and through the array of channels into the subsequentcompartment, the relative volume fraction of WBCs, RBCs, or otherparticles is increased in the first compartment. The two streams, onecontaining substantially undiluted blood or particles, and the other, aliquid-only fraction, recombine in the subsequent compartment to form aliquid sample having a reduced volume fraction of cells or particles inthe subsequent compartment. A further benefit of the invention is toprovide a thickness within the chamber during manufacture, which is moreaccurate than would otherwise be obtained since the wall surrounding theinterior of the chamber acts to hold the two opposing sample containmentwalls, that function as optical windows apart at a fixed and accuratedistance.

FIG. 1 is merely exemplary and is not intended to limit the presentinvention in any way. A separation chamber in accordance with thepresent invention can have notches in one or more compartments of theseparation chamber, or no notches at all.

In the embodiment of the present invention demonstrated in FIG. 1, theseparation chamber is surrounded by a wall (1) which is used to carry anoptically transparent lid (not shown) allowing for optical observationand to contain the liquid sample. The separation chamber is divided intotwo compartments by a separation wall (2) which has separation channelson top of it. Details of different embodiments for separation wall (2)are shown in FIGS. 2, 3, and 4. Returning to FIG. 1, fluid fills thefirst compartment through a sample entrance (3), which is surrounded byan extension (11) of wall (1), and moves into the first compartment (4)of the chamber. Upon proceeding, the liquid encounters a first notch (5)displaced laterally across the flow path. The notch creates a momentarybarrier to progression of the advancing fluid meniscus until themeniscus has contacted the notch across the whole width of the chamber.Eventually the fluid wets into the notch, allowing the fluid to advanceto the next notch (6) where the meniscus is again evened out. The fluidadvance continues in this manner until it reaches the separation wall(2). The fluid continues to advance through the separation wall andseparation channels to the second compartment (7) until finally stoppingat its far end (8). Adequate venting of air while the chamber fills withliquid is provided by a series of venting-channels (13) on top of wall(1). To allow for a free passage of air out of channels (13), walls (1)and (11) are surrounded by a moat (12). During the filling process, theparticles of interest, are prevented from passing through the separationchannels that traverse separation wall (2), and as a consequence,concentrate in the first compartment (4). After the fluid fills thesubsequent first and the second compartment it becomes quiescent,allowing optical examination.

FIG. 2 shows a separation wall having two different channels ofdifferent sizes (9) and (10). The channel labeled (9) is sized to allowonly particles smaller than a certain size to pass and the channellabeled (10) is sized to allow only liquid to pass. It has been foundthrough experimentation that these channels can be sized appropriatelyto exclude cell or particle sizes of interest.

FIG. 3 shows a separation wall (16) with a plurality of one type ofseparating channel (17) disposed laterally across its full width.

FIG. 4 shows another embodiment of the separating channels, with aplurality of a first channel (14) which allows cellular or particulatespecies to pass through it, while allowing a liquid portion devoid ofthis species to pass through a plurality of a second channel (15)disposed in an array alongside the first channel (14).

In another embodiment of the present invention, which is depicted inFIG. 5, increased analytical sensitivity can be attained by enlargingthe volume of the second compartment, allowing the sample portion in thefirst compartment to accumulate larger cells over an increased volume.

In the embodiment of FIG. 5, the separation chamber is surrounded by awall (21) which is again used to carry an optically transparent lid (notshown) allowing for optical observation and to contain the liquidsample. The separation chamber is divided into two compartments (22) and(23) by a separation wall (24) which has the separation channels on topof it. Separation wall (24) is U-shaped in order to make it as long aspossible. In operation, fluid fills the first compartment (22) through asample entrance (25), which is surrounded by an extension (27) of wall(21), and moves into the first compartment (22) of the chamber. Uponproceeding, the liquid encounters a first notch (25) displaced laterallyacross the flow path. The notch creates a momentary barrier toprogression of the advancing fluid meniscus until the meniscus hascontacted the notch across the whole width of the chamber. Eventuallythe fluid wets into the notch, allowing the fluid to advance to the nextnotch (26) where the meniscus is again evened out. The fluid advancecontinues in this manner until it reaches separation wall (24). Thefluid continues to advance through separation wall (24) and theseparation channels to the second compartment (23) which is surroundedby wall extension (28). Wall (21) and wall extension (28) are equippedwith air-venting channels (29) and (30), respectively. To allow for afree passage of air out of channels (29) and (30), walls (21), (27), and(28) are surrounded by a moat (31) in base plate (32). The lid isresting on walls (21), (27), and (28).

During the filling process, the particles of interest are prevented frompassing through the separation channels that traverse separation wall(24), and as a consequence, concentrate in the first compartment in aU-shaped area (33) in front of separation wall (24). As mentioned above,compartment (23) behind separation wall (24) has an increased volume,which, in connection with the elongated U-shaped separation wall (24)allows one to process a larger sample volume, as compared to theembodiments shown in FIGS. 1, 2, 3, and 4. In other words, theembodiment of FIG. 5 is suitable to achieve reasonable particleconcentrations for analysis in front of separation wall (24), even ifthe particle concentration in the incoming sample is rather low.

What is claimed is:
 1. An apparatus for obtaining a liquid sample havingan increased cellular or particulate concentration for opticalexamination, comprising: a separation chamber; a wall surrounding saidseparation chamber; a separation wall in said separation chamberdividing said chamber into first and second compartments, saidseparation wall having separation channels of at least two differentsizes traversing said separation wall; a sample entrance into said firstcompartment for receiving sample fluid; air-venting channels on top ofsaid wall surrounding said separation chamber; and a flow path in saidseparation chamber, wherein said flow path is in flow communication withthe sample entrance and said flow path flows from said sample entrancethrough the first compartment and flows through the separation channelsin the separation wall into the second compartment.
 2. The apparatusaccording to claim 1 wherein said liquid sample is blood.
 3. Theapparatus according to claim 1 wherein said apparatus further comprisesa lid portion.
 4. The apparatus according to claim 3 wherein said lidportion has at least one opening for sample delivery.
 5. The apparatusaccording to claim 1 wherein said separation channel of a first size isabout 3 to 10 um deep by 5 to 50 um wide.
 6. The apparatus according toclaim 1 wherein said separation channel of a second size is about 0.5 to1.5 um deep by 50 to 1000 um wide.
 7. The apparatus according to claim 1wherein said apparatus further comprises a plurality of notches in saidseparation chamber.
 8. The apparatus according to claim 1 wherein saidapparatus further comprises a plurality of notches in said firstcompartment of said separation chamber.
 9. The apparatus according toclaim 1 wherein said apparatus further comprises a plurality of notchesin said second compartment of said separation chamber.
 10. The apparatusaccording to claim 1 wherein said apparatus further comprises aplurality of notches in said first compartment and second compartment ofsaid separation chamber.
 11. The apparatus according to claim 1 whereinsaid separation wall has a plurality of separation channels of the samesize.
 12. The apparatus according to claim 1 wherein said separationwall has a plurality of separation channels of different sizes.